DESCRIPTION: (provided by the applicant): The cyst wall of Giardia lamblia is critical to its success as a parasite. This primitive extracellular matrix is responsible for giardial survival in the environment and for its ability to infect new hosts. Based on our extensive studies of giardial encystation, we have formulated a four-stage model of cyst wall protein (CWP) and cyst maturation. The goal of this revised proposal is to validate and expand that model for a more comprehensive understanding of this essential differentiation. We will focus in depth on CWP processing, transport, and assembly. Our central hypothesis is that assembly of the cyst wall requires co-coordinated activities within several intracellular compartments, but final maturation occurs on the cell surface. Specific Aim 1 is to evaluate the role of the novel giardial lysosomal/endosomal peripheral vesicle (PV) compartment in processing of CWPs. We will test the hypothesis that passage through the PV is necessary for CWP maturation. Alternatively, CWP may traffic through the PV in an endocytic pathway to recover CWP that has been released prematurely. We will use tetracycline-regulated expression of epitope-tagged CWPs and cell surface labeling to trace CWP. These studies will elucidate the role of CWP traffic through the PV in achieving mature cyst wall architecture and function. In Specific Aim 2, we will test the hypothesis that CWPs undergo sequential stages of S-S bond formation and re-arrangement catalyzed by unusual giardial protein disulfide isomerases, leading to formation of resistant fibrils on the cell surface. Specific Aim 3 is to test the hypothesis that iso-peptide bond formation is needed for differentiation into cysts and to identify the proteins cross linked, using specific labels and inhibitors. This revised proposal will reveal basic insights into formation, function, and evolution of an ancient eukaryotic extracellular matrix, using integrated biochemical, and cell biological, and molecular approaches. Since cystic stages are key to transmission of many parasites, cyst wall formation is a target for interruption of the life cycle.